Clostridium neurotoxin includes Clostridium botulinum neurotoxin and Clostridium tetanus toxin.
Clostridium botulinum neurotoxins (hereinafter referred to botulinum toxin) is a group of proteins known to have the highest toxicity (including types A-G), and is produced primarily by Clostridium botulinum in anaerobic condition. It mainly inhibits nerve ending from releasing acetylcholine, causing muscle relaxation and paralysis, especially paralysis in respiratory muscles as a major cause of death. For type A botulinum toxin in aerosol, the lethal dose of inhalation to human is 0.3 μg, LCt50 is 0.1-0.5 mg·min/m3, lethal dose of intravenous injection is 0.15-0.3 μg, and lethal dose of oral administration is 8-10 μg. Botulism may happen in many ways, including when botulinum toxin aerosol released in an environment (botulinum toxin exposure), causing respiratory inhalation poisoning. Due to technical limitations, releasing aerosol in a large scale does not happen easily. Currently, the main routes of poisoning are food contamination and contracting toxin by water passing through the digestive tract. Occasionally, would infection leads to poisoning (botulinum toxin poisoning). In addition, type A botulinum toxin has been widely used in the clinical treatment of cholinergic nerve and muscle dysfunction, as well as to eliminate wrinkles in cosmetic practices. During use in treatment, botulism symptoms may occur due to overdose, misuse, abuse, and/or adverse reaction. Botulism have relatively short incubation period, fast progression, relatively severe state of illness, and high mortality. Poisoning in human is mainly caused by botulinum toxin of types A, B, E, and F, in that types A and B have the highest neurotoxicity to human.
Clostridium tetanus toxin is produced by Clostridium tetani in anaerobic condition, which has high affinity to spinal anterior horn cells and brain stem cells, and could prevent releasing of inhibitory neurotransmitter from presynaptic membrane, thereby causing symptoms such as intense muscle spasm, cardiac arrhythmia and respiratory failure, etc. Wound infection is the main way to cause tetanus, when the body sustains a trauma and the wound is contaminated, Clostridium tetani can invade local wound surface causing exogenous infection, and with bacteria propagating at local site, releasing toxin, and causing tetanus.
Currently, antitoxin horse serum is used for prevention and treatment of exposure to Botulinum Neurotoxins or Tetanus toxin, botulism and tetanus. It is effective for more than 80% of poisoned patients. However, this type of medicine has significant side effects, with severe hypersensitivity reaction (including anaphylactic and anaphylactoid reactions) and delayed anaphylactic reaction (serum sickness) possibly occurring after administration of the antitoxin. Clinical reports show about 9% of cases experience serum sickness and anaphylactic reaction. In addition, antitoxin is produced from horse plasma, which may have the risk of carrying pathogens such as virus. These disadvantages limit wide application of antitoxin horse serum, and raise demand for development of new type of medicine.
Clostridium botulinum neurotoxin and Clostridium tetanus toxin share similar pathogenic process in that the C-terminus of heavy chain of toxin binds to the ganglioside on the membrane of the nerve cells, rearranges its structure in acidic environment, and promotes N-terminus of heavy chain entering into membrane. Meanwhile, the light chain unfolds and the disulfide bond is reduced and transferred into cells as zinc ion metalloenzyme, catalytically cracking a class of intracellular substrate proteins (types A and E Clostridium botulinum neurotoxin act on synaptosome associated protein SNAP-25, while types B, D, F, and G Clostridium botulinum neurotoxin and Clostridium tetanus toxin act on synaptic vesicle associated membrane protein VAMP), thereby affecting the transfer of acetylcholine and intervening conduction of nerve impulse, so as to cause paralysis or excitement of motor nerve. If one or more aspects of binding, cytosis and catalysis is/are inhibited or repressed, the neurotoxicity of toxin can be suppressed effectively. Targeting design of light chain of toxin having enzymatic activity and development of inhibitor are the hot spot of researches recently.
Harry B et al. performed in vitro high-throughput screening of natural extract from plants, marine tissue, and fungus for substance inhibiting clostridium toxin, found 30 non-subtype inhibitors, of which 5 extracts can suppressed both types B and E botulinum toxin. Anne C et al. developed a class of highly reactive sulfur-containing peptide compounds, wherein Ki value of the compound with the highest activity to light chain of type B botulinum toxin was up to 2.3 nM, but follow-up report on overall activity in animals is not seen (Reference 1: Anne C, et al. J Med Chem, 2003, 46:4648-4656). Salzameda N T et al. 2011 found, L-cichoric acid significantly inhibited light chain of type B botulinum toxin, and the compound had certain inhibitory activity on subtype A (reference 2: Salzameda N T, et al. Chem Commun, 2011, 47:1713-1715), the activity was relatively weak. Pang, Y.-P. et al. 2009 found that AHP, an azole-based compound, was a potential inhibitor, which could effectively neutralize toxicity of type A botulinum toxin on N2a cells through binding to Zn catalytic region in the large hydrophobic region pocket of the active site of type A botulinum toxin light chain (reference 3: Pang, Y.-P. et al. PLos One 2009, 4, e7730.), but still no in vivo activity has been reported. Eubanks, L. M. et al. 2010 found that lomofungin could inhibit light chain of type A botulinum toxin (Ki value 6.7±0.7 uM), showing a typical non-competitive dynamics, but the compound had multiple unstable phenolic hydroxyl groups and aldehyde groups (reference 4: Eubanks, L. M., et al. ACS Med. Chem. Lett. 2010, 1:268-272.).
Focusing on the shortcomings of the inhibiting compound candidates that have been developed, there is a need to develop a class of active compounds which potentially have wider spectrum of in vitro inhibition and antagonism against clostridium neurotoxin (multiple types of botulinum toxin and clostridium tetanus toxin), and which can pose a higher anti-poisoning activity among all animal species, while providing better pharmaceutical efficacy, from which new medicine can be developed therefrom.